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Biological nitrogen fixation in maize (Zea mays L.) by 15N isotope-dilution and identification of associated culturable diazotrophs

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Abstract

The nitrogen-fixing capacity of a range of commercial cultivars of maize (Zea mays L.) was evaluated by the 15N isotope-dilution method. Biological nitrogen fixation (BNF) expressed as percent nitrogen derived from air (Ndfa) ranged from 12 to 33 regardless of nitrogen fertilization. BNF was not affected by mineral nitrogen fertilization except on cultivar Topacio and PAU-871 cultivars. Subsequently, culturable bacterial diazotrophs were isolated from endophytic tissue of maize: seed, root, stem, and leaf. All isolates were able to grow on N-free semisolid medium. Eleven bacteria isolates showed nitrogen-fixing capacity by the reduction of acetylene to ethylene and confirmed by PCR the presence of nifH gene in their genome. Identification of the 11 isolates was performed by bacteriological methods, 16S rRNA gene sequences, and phylogenetic analysis, which indicated that the bacteria isolated were closely related to Pantoea, Pseudomonas, Rhanella, Herbaspirillum, Azospirillum, Rhizobium (Agrobacterium), and Brevundimonas. This study demonstrated that maize cultivars obtain significant nitrogen from BNF, varying by maize cultivar and nitrogen fertilization level. The endophytic diazotrophic bacteria isolated from root, stem, and leaf tissues of maize cultivars may contribute to BNF in these plants.

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Acknowledgment

This work was supported by the International Atomic Energy Agency. We would like to thank Ivanna Taramasco for assistance with the phylogenetic analysis, Claudia Barlocco for technical assistance, and Christine Lucas for review of English.

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  1. Laboratorio de Microbiología del Suelo, Centro de Investigaciones Nucleares (CIN). Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Iguá 4225, CP 11400, Montevideo, Uruguay

    Adriana Montañez, Cecilia Abreu & Margarita Sicardi

  2. Laboratorio de Tecnología Molecular, Facultad de Ciencias, Universidad de la República Oriental del Uruguay, Iguá 4225, CP 11400, Montevideo, Uruguay

    Paul R. Gill

  3. Laboratorio de Ecología Microbiana, Departamento de Bioquímica, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, Montevideo, Uruguay

    Adriana Montañez & Paul R. Gill

  4. Soil Science Unit, FAO/IAEA, Agency’s Laboratories Seibersdorf, A1400, Vienna, Austria

    Gudni Hardarson

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  1. Adriana Montañez
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Montañez, A., Abreu, C., Gill, P.R. et al. Biological nitrogen fixation in maize (Zea mays L.) by 15N isotope-dilution and identification of associated culturable diazotrophs. Biol Fertil Soils 45, 253–263 (2009). https://doi.org/10.1007/s00374-008-0322-2

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